The long-term goals of this project are to use genetic approaches to investigate the functions of opioid receptors. Three distinct opioid receptor cDNAs have recently been cloned that represent the major receptor classes previously defined pharmacologically. The availability of these probes thus provides a unique opportunity to apply molecular approaches, in combination with biochemical, behavioral and cellular methods, to examine the roles of endogenous opioids in development, pain perception and neuroimmune function. Experiments addressing three specific aims are proposed. In the first, we will determine the ontogeny of the delta, mu, and kappa opioid receptors using in situ hybridization and immunocytochemistry. The distribution and timing of receptor transcript and protein expression will be investigated and compared to existing binding data and to the expression patterns of endogenous ligands including POMC, enkephalin, and dynorphin. These studies are designed to identify putative sites of endogenous opioid action during development, to determine the specific receptor classes active at those sites, and provide a basis for examining potential developmental defects caused by receptor inactivation. In the second aim, we will use gene targeting procedures to inactivate the delta, mu, and kappa opioid receptor genes. We will isolate mouse opioid receptor genomic clones and use the clones to construct targeting vectors containing selectable markers. We will apply methods, already successfully used in our laboratory, to substitute one opioid receptor null allele for one wild-type allele in embryonic stem cells. These cells will be injected into blastocysts and germ-line chimeras will be identified, which will allow us to generate new animal strains lacking opioid receptors. These strains will be examined for developmental abnormalities at the sites of normal receptor expression identified in Aim 1 and mated to produce strains with multiple mutant receptors. In addition, we will use radioligand binding assays and assays that differentiate in vivo each receptor subtype to determine whether, in an individual knock-out, all subtypes of each receptor class are coordinately lost. We will also compare the patterns of expression of non-mutated opioid receptor genes in specific receptor mutants with the normal patterns to determine whether compensation at the transcriptional level accompanies any of the three mutations. In the third aim, we will examine the behavioral and physiological consequences of receptor inactivation using two well-established experimental models. First, a series of behavioral assays of opioid function will be used to monitor the effect of receptor inactivation on pain perception. These studies will focus on stress-induced analgesia and the analgesic actions of opiate drugs such as morphine. In the second, we will investigate a newly discovered role of endogenous opioids in regulating macrophage chemotaxis after injury to the developing brain. We will analyze the migratory behavior of brain phagocytes from normal and mutant mice following visual cortex ablation using markers for macrophages and microglia and in vitro assays of chemotaxis. Taken together, these studies should establish clear roles for each opioid receptor class in specific biological processes associated with behavior and drug addiction.